Cystic fibrosis (CF) is an autosomal recessive genetic disease of the secretory glands that mainly affects the lungs, pancreas, intestine and liver. Cystic fibrosis, also known as mucoviscidosis, is the most common fatal recessive genetic disease in northern countries.
Cystic fibrosis is associated with mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene, which encodes a cyclic-AMP dependent chloride channel at the apical membrane of epithelial cells. This channel mediates the transport of specific anions (e.g., chloride and thiocyanate) against their electrochemical gradient and hence acts to regulate the water content and the ionic composition of sweat, digestive juices and bronchial mucus.
The gene encoding CFTR has been identified and sequenced (See Gregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature 347:358-362; Riordan, J. R. et al. (1989) Science 245:1066-1073).
Mutations affecting the function of the CFTR gene cause a major imbalance in ion and fluid transport across the epithelial cell membrane, most importantly at mucosal surfaces. In the lungs, the resulting decrease in chloride transport contributes to enhanced mucus dehydration and defects in mucocilliary clearance, leading to mucus accumulation, bacterial colonization and inflammation. This results in recurrent respiratory tract microbial infections that are the primary cause for morbidity and mortality in cystic fibrosis patients. CFTR loss-of-function also causes imbalances in ion and fluid transport in other major exocrine glands. Consequently, CF patients suffer from gastrointestinal symptoms and pancreatic insufficiency that have to be compensated for by a proper diet and can also, if left untreated, result in death. The majority of males with CF are infertile and fertility is decreased among CF females.
Sequence analysis of the CFTR gene of CF patients' chromosomes has revealed a variety of disease-causing mutations. To date, more than 1000 disease-causing mutations in the CFTR gene have been identified. The most common CFTR mutant (in about 70% of CF patients) consists of the deletion of phenylalanine at position 508. The deletion of residue 508 in ΔF508-CFTR induces major folding defects that primarily result in the inability of the CFTRΔF508 protein to reach its proper location at the plasma membrane due both to increased endoplasmic reticulum (ER) retention and ER-associated degradation (ERAD) by the ubiquitin-proteasome machinery. The marginal targeting of the partially functional ΔF508-CFTR protein at the plasma membrane is not sufficient functional and therefore assimilates to a loss of function. Artificially increasing the number of CFTR channels targeted to the membrane showed that CFTRΔF508 remains partially functional although the ΔF508 mutation exhibits an intrinsic channel gating defect and accrued protein turnover at the plasma membrane. The folding defects of CFTR imposed by the ΔF508 mutation drastically reduce the number of functional channels reaching the apical membrane and thereby anion and fluid transport across epithelia (Ward and Kopito, 1994, J. Biol. Chem. 269, 25710-25718; Ward and Kopito, 1995, Cell 83, 121-127; Lukacs et al., 1994, EMBO J. 13, 6076-6086; J. R. Riordan, Am. J. Hum. Genet. 1999 June, 64(6):1499-1504. PMCID: PMC1377893).
Cystic fibrosis mutations impact CFTR function through different mechanisms that must be considered when designing therapeutic strategies. Class II mutations (in 88% of CF patients) include the above-mentioned most prevalent mutation, CFTRΔF508; these mutations prevent ER folding of CFTR, and hence its trafficking in the secretory pathway, and trigger degradation of the mutant proteins by ERAD.
Both gene therapy and pharmacotherapy have been proposed as ways to restore CFTR function, but these are currently lacking for class II mutants.
Treatment directed at class II mutations aims at increasing the amount of proteins at the plasma membrane by correcting their trafficking defect. CFTRΔF508 also displays reduced channel opening and increased turnover when forced at the plasma membrane. Therefore, efficient restoration of CFTR-class II mutation function might also require molecules aimed at correcting the gating defects of these mutations and at increasing plasma membrane stability, in addition to the interventions aimed at increasing protein plasma membrane expression. Such molecules are already available (VX-770 and VX-809, respectively) (Van Goor et al., PNAS 2009; Van Goor et al., PNAS 2011). Compounds aimed at restoring CFTR trafficking are defined as “correctors”, those improving the channel opening are named as “potentiators”, and a combined therapy including correctors and potentiators is currently considered for CFTRΔF508 patients. Correctors can be separated into two classes: those that bind to CFTR molecules and act on its folding rate and stability, and are therefore named “pharmacological chaperones”, and those that act on the proteostasis network, i.e., that target proteins that regulate CFTR folding, degradation and/or vesicular trafficking (proteostasis regulators (PR)). However, currently identified correctors remain poorly effective on their own and their mode of action and CFTR specificity remain poorly defined. Recent trends points to the use of a combination of correctors that target CFTR folding defects occurring during biosynthesis to enhance CFTR maturation and plasma membrane expression (Okiyoneda and Lukacs (2013) Nature Chem. Biol.).
Inhibition of the Endoplasmic-Reticulum-Associated Degradation (ERAD) machinery promotes CFTR stabilization in the endoplasmic reticulum (ER); components of ERAD have been proposed to be relevant PR targets in CF treatment (Younger et al. (2006) Cell 126, 571-582; Younger et al. (2004) J. Cell. Biol. 167, 1075-1085). Inhibition of ERAD is already considered beneficial by increasing the amount of mutant CFTR made available to chemical chaperones, if not by enhancing CFTR plasma membrane expression.
General ERAD inhibition as performed by proteosomal inhibition is not well-suited, as it results in the accumulation of insoluble ubiquitinated aggregates, hampering further export of the protein out of the ER.
E3 ligases are central components of the ERAD machinery, tagging ERAD substrates for subsequent degradation by the proteasome. ERAD E3 ligases are targets that are considered suitable to inhibit CFTR degradation, as: i) their inhibition is believed to stabilize ERAD substrates in a soluble state; ii) they are relatively substrate-specific; and iii) their function should be conserved in many cell types. E3 ligases' specificity is dictated by their association with specific chaperones that operate by recruiting the E3 ligases to their specific substrates. Identification of these chaperones and of their interaction interfaces should help define drug target domains in ligases to further increase specificity. The development of efficient E3 ligase inhibitors has been validated as a therapeutic strategy in CF.
So far, two E3 ligases, RNF5 and CHIP, have been associated with the ER degradation of CFTR proteins, more specifically CFTRΔF508. Inactivation of RNF5 or CHIP promotes the stabilization of the mutant protein in a foldable state and an increased maturation of the mutant protein when cells are co-treated with a pharmacological chaperone (e.g., the Corr-4a corrector). In this specific case, Corr-4a and RNF5 depletion target distinct checkpoints of the CFTR quality control; RNF5 targets the first checkpoint, which occurs during CFTR synthesis. RNF5 is thought of as a major target for increasing the amount of foldable CFTR proteins (Younger et al. (2006) Cell 126, 571-582; Grove et al. (2009) Mol. Biol. Cell 20, 4059-4069).
There is at present no efficient cure for cystic fibrosis. Current treatments are only palliative: they include antibiotic cures (for lung infections), chest physiotherapy/mechanical expectoration (for mucus accumulation), surgery and mechanical ventilation. Hence there is a strong need for the development of new therapies for the treatment of cystic fibrosis.